1. Field of the Invention
The present invention relates to a one-stage process for the resalting and purification of oligopeptides.
2. Background Information
Oligopeptides frequently display biological activity and are therefore used as therapeutic agents. LHRH agonists and antagonists may be mentioned by way of example, which are used, inter alia, to treat certain types of cancer.
The oligopeptides to be purified may be prepared according to processes known in the prior art. Suitable processes include, among others, the Merrifield peptide synthesis on solid support materials or the conventional synthesis in solution. Both in the Merrifield solid phase synthesis and in synthesis in solution it is essential to provide certain regions in the molecule with protective groups that are split off at the end of the preparation. In the solid phase synthesis it is moreover necessary to remove the oligopeptide from the solid support. For further details of the synthesis of peptides reference may be made to the relevant literature (Houben-Weyl, Methoden der organischen Chemie, Vol. 15/1 and 15/2; M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag 1984).
If the peptide to be prepared is a pharmaceutical, then it is often desirable for the oligopeptide to be present in the form of its acetate salt in order not to have to give the patient any foreign or other potentially harmful substances in conjunction with the administration of the drug.
It is often the case, however, that the oligopeptide, owing to circumstances connected with the synthesis, does not necessarily exist in the form of the acetate salt, either because acids other than acetic acid have to be used for the final cleavage of the protective groups, or because the free form of the peptide cannot be prepared or can be prepared only with difficulty and it is not possible to perform a simple conversion to the acetate by means of acetic acid. In order to cleave the protective groups or to cleave the peptide from the resin required for the synthesis, recourse generally has to be made to relatively strong acids such as trifluoroacetic acid, hydrochloric acid or hydrobromic acid. For further details of these cleavage processes reference may be made once again to the standard textbooks (Houben-Weyl, Methoden der organischen Chemie, Band 15/1 and 15/2; M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag 1984).
In order to prepare the required acetate of the relevant oligopeptide for use in animals or humans, one is required in the aforementioned cases to resalt the oligopeptide.
The oligopeptide to be tested as active substance, or that is already available commercially as a therapeutic agent, must satisfy particular requirements as regards its purity. Because of the lack of a suitable conventional purification method, the product mixture formed in the synthesis is generally purified by means of chromatography, in particular high pressure liquid chromatography. For this purpose the oligopeptide must be taken up in a solvent, preferably in the solvent mixture of the mobile solvent chosen as eluent, before it is applied to the column.
For oligopeptides several processes have previously been described in the literature that relate to their resalting and purification. According to Gabriel (Int. J. Peptide Protein Res. 1987, 30, 40-43) the oligopeptide GRF (1-44) --NH.sub.2 can be converted from its trifluoroacetate into the acetate by means of high pressure liquid chromatography using pyridine-containing and acetic acid-containing solvents. With regard to the pyridine residues that inevitably remain in the oligopeptide after such a procedure, there is concern, of course, about the toxicological properties of this substance. Also, a purification process that involves relatively large amounts of dangerous pyridine is undesirable from industrial safety aspects.
Hoeger et al. (Biochromatography 1987, 2, 134-142) have attempted to resalt and purify GnRH peptides while avoiding the use of a pyridine-containing solvent system. Starting from the fluoride salts, a two-stage reversed phase gradient chromatography was carried out in triethylammonium phosphate (TEAP) and trifluoroacetate (TFA) buffers with acetonitrile as modifier. Lyophilisation of the purified peptide fractions was followed by conversion to the acetate salts via anion exchange chromatography with dilute acetic acid or reversed phase chromatography in an ammonium acetate/acetonitrile gradient.
EP 0145258 describes, inter alia, the purification of HF salts of nonapeptides and decapeptides of the group of LHRH agonists. Here, too, the resalting takes place separately from the purification step, first via anion exchange chromatography followed by final purification on an octadecyl-silanised silicic gel phase by means of an eluent consisting of ammonium acetate and acetonitrile under high pressure conditions.